Bioinformatics for DNA-seq and RNA-seq
experiments
Li-San Wang Department of Pathology and Laboratory Medicine Penn Institute for Biomedical Informatics Penn Genome Frontiers Institute University of Pennsylvania Perelman School of Medicine
Next Generation Sequencing Technology
Generate reads of billions of short DNA sequences in the order of 100nts in a week
Costs < $5K for resequencing a human genome
Hi-Seq 2000: run 2 flow cells (300Gb each) in ~ 1 week, sequences 6 genomes
Illumina Hi-Seq 2000
Applications of NGS DNA-Seq resequences genomes to identify
variations associated with diseases and traits Use RNA-Seq to study gene expression
activities Use ChIP-Seq and DNase-Seq to measure
protein-DNA interactions and modifications … Many other types of protocols
Central Dogma DNA
RNA
Protein
Phenotypes
RNA-Seq
RNA
Library prep
Sequencing andAnalysisImages: illumina
Reverse Transcription & DNA fragmentation
High read heterogeneity along RNA transcripts
Needs to dig deeper! Secondary structures Functional classes Modifications (non-
standard nucleotides) Visualization … and many other
questions
SAVoR: RNA-seq visualization Fan Li, Paul Ryvkin, Micah Childress, Otto Valladares, Brian Gregory*, Li-San Wang*. SAVoR: a server for sequencing annotation and visualization of RNA structures. Nucleic Acids Research, 2012.
HAMR: Detect RNA modification using RNA-seqPaul Ryvkin, Yuk Yee Leung, Micah Childress, Otto Valladares, Isabelle Dragomir, Brian Gregory*, and Li-San Wang*. HAMR: High throughput Annotation of Modified Ribonucleotides. RNA, in press, 2013.
CoRAL: Use small RNA-seq to annotate non-coding RNA function classesYuk Yee Leung, Paul Ryvkin, Lyle Ungar, Brian Gregory*, Li-San Wang*. CoRAL: Predicting non-coding RNAs from small RNA-sequencing data. Nucleic Acids Research, 2013.
RNA-Seq-Fold: Use pairing-informative RNA-seq protocols to estimate secondary structures (in progress)CoRAL
SAVoR: web-based visualization of RNA-seq data in a structural context
http://tesla.pcbi.upenn.edu/savor/
RNA-seq data +2nd structure= SAVoR Plots !
Li et al., NAR 2012
Log-ratio of dsRNA-seq to ssRNA-seq read coverage along the At2g04390.1 transcript.
Modified RNA – Motivation:Sites with unusual mismatch patterns in RNA-seq
1. A in actual sequence, C/G/T are due to 1% base calling error rate
2. A/C SNP, G/T are due to 1% error rate3. G/T ratio too far away from 1:1, heterozygotes
cannot explaina. A and C rates are too high for base calling
error
123
3a
Observed nucleotide pattern at a known m2G siteIn an Alanine tRNA
N-2-methylguanosine (m2G)
guanosine (G)
H2
N
12 3 4
56 78
912 4
56 78
93
3' 2'
5'
3' 2'
5'
tRNA-modifying protein
Watson-Crick pairing edge has been modified
tRNA modifications
Watson-Crick edge
Detecting modified RNAs: change in RT effects when Watson-Crick edge is modified
Statistical model for HAMR
H01: homozygous reference, low base calling error
H02: heterozygote, low base calling error
In both cases, there should be at most two nucleotides with high frequencies
ML ratio test Annotation: naïve Bayes model on non-reference allele frequencies
Results
Statistical analysis on known modification sites show this idea works with high specificity
Known modificationspredicted to affect RT
Detected modificationspredicted to affect RT
Our data
Yeast dataset
Precursor Classes Observations Accuracy
A m1A|m1I|ms2i6A, i6A|t6A 187 98%
G m1G, m2G|m22G 86 79%
U D, Y 17 96%
Train on human tRNA data, test on yeast tRNA data
Classification accuracy
Modifications in other RNAs Scan the entire smRNA transcriptome for candidate
modified sites
* Uniquely mapped reads in 4 libraries
* Removed sites corresponding to read-ends
* Removed sites corresponding to known SNPs
HAMR High-Throughput Annotation of Modified RNAs Ryvkin et al., RNA, 2013 http://tesla.pcbi.upenn.edu/hamr/ Please contact us if you are interested!
RNA-seq is more than an expensive digital gene expression microarray
NGS algorithms and experimental protocols should integrate tightly
Bioinformatics scientists
Bench scientists
DNA-Seq: find genetic variations linked to traits and diseases
All individuals have small differences between each other Single nucleotide polymorphism
(SNP) is the most common form Other types: indel, copy number
variation, rearrangement Genetic polymorphisms may lead
to different phenotypes and diseases 21 trisomy: Down syndrome Substitution 1624G>T of the CFTR gene
leads to change of amino acid (G542X) which leads to cystic fibrosis
Alzheimer’s Disease Sequencing Project Announced in Feb. 2012 Participants
NIA, NHGRI ADGC and CHARGE Large-Scale Genome Sequencing and Analysis Centers
(Broad/Baylor/WashU) NACC (phenotype) and NCRAD (sample) NIAGADS (data coordinating center) NCBI dbGaP/SRA
Design: 584 WGS / 11,000 WES (>300TB data) WGS data of 584 samples available from our ADSP data
portal Visit ADSP website www.niagads.org/adsp to learn about
study design, apply for data access, download dataPhoto from http://nihrecord.od.nih.gov/newsletters/2012/03_02_2012/story5.htm
Computational Challenges to Analyzing DNA-Seq data Mapping between 100~1000 billion reads to the
reference genome with good sensitivity Variant calling: call SNPs and structural variants reliably Association: Find susceptibility variants by association
tests Interpretation: Interpret the effect of variants Data management: Query, store, and distribute 100TBs
of data
~~ And that’s just for one project!
Cloud computing using Amazon EC2 Can run hundreds of cores on Amazon EC2 easily Can share data and programs easily Very good security Steep learning curve
Needs to provide pre-configured workflows/environments allows you to run analysis easily on Amazon
Storing data is very expensive $0.1/GB-Month, or $1200/TB-year Glacier is 10 times cheaper but also that much slower
DNA Resequencing Analysis Workflow (DRAW)
Easy to run – invoke phases by five commands, no need to mouse-click like crazy
Memory request based on data size Support SunGridEngine for cluster
computing Modular architecture, job
monitoring, job dependency, auditing, error checking
Runs on Amazon EC2, $582/FC We are migrating all our NGS
pipelines to DRAW architecture
Mapping
Realignment, dedup,
uniq, base quality
recalibration
Variant detection
Coverage, QC
metrics
BWA
GATKPicardSamtools
GATKGATKSamtools
NIA Genetics of Alzheimer’s Disease Data Storage Site (NIAGADS)
Portal to AD genetics studies funded by NIA
Portal for ADSP data Portal for other large-scale AD
sequencing projects (>2,000 whole genomes, >400TB raw data) being developed
Software (DRAW+SneakPeek) and other resources
Signup for user account and news alert at
www.niagads.org
Lab members
Mugdha Khaladkar Dan Laufer
Chiao-Feng Lin Otto Valladares
Fan Li
Micah Childress
Fanny Leung
Yih-Chi Hwang
Paul Ryvkin
Amanda PartchTianyan Hu
Mitchell Tang
John Malamon
Alex Amlie-Wolf Pavel Kuksa
AcknowledgementsPathology and Lab Medicine
PSOM/CHOPDavid RothNancy SpinnerDimitrios MonosJennifer MorrisetteRobert DaberLaura ConlinEllen TsaiAvni SantaniZissimos Mourelatos
Support:Penn Institute on AgingPGFIAlzheimer’s FoundationCurePSP foundationNIH: NIA/NIGMS/NIMH/NHGRI
Schllenberg labGerard SchellenbergEvan GellerLaura Cantwell
Gregory LabBrian GregoryQi ZhengIsabelle DragomirJamie YangSandeep Jain
CNDR/ADCJohn TrojanowskiVirginia LeeVivianna Van DeerlinSteven ArnoldTerry SchuckRobert Greene
Maja BucanChris StoeckertArupa GangulyKate NathansonAlice Chen-PlotkinTravis Unger
Mingyao LiJohn HogeneschNancy ZhangSampath KannanLyle UngarSarah Tishkoff